Tag: microbiology

The patient is a 31 year old man with a history of intravenous drug use with last reported use nine months previous, who reports low back pain. The patient’s symptoms started as a mild pain and progressively worsened over two weeks to the point that he was unable to stand or ambulate. He also developed intermittent radiation of pain to the bilateral lower extremities and associated symptoms of chills and diaphoresis. Blood cultures were sent. MRI showed an epidural abscess at the level of L5-S1. The patient underwent lumbar spinal decompression surgery, and intra-operative cultures were sent for evaluation.

Serratia marcescens is a motile, facultatively anaerobic, gram negative bacillus of the Enterobacteriaciae family. Some strains of Serratia produce a distinctive brick red pigment, prodigiosin (Image 4), although non pigmented strains are frequently isolated from human infection sites. Serratia marcescens is one of the few Enterobacteriacea that produces DNAse, lipase, and gelatinase. It does not usually ferment lactose. This species is widely present in the environment, including in animals, insects, plants, water, and soil, but unlike other Enterobacteriaciae species it is not a typical component of normal human fecal flora.

Eight species of Serratia have been found to cause infections in humans. Of these, >90% are caused by Serratia marcescens (1). This is a rare cause of infection in immunocompetent hosts but can cause opportunistic nosocomial infections, especially following invasive procedures such as such as intravenous catheterization, respiratory intubation, and urinary tract manipulations. The most common infections caused by Serratia marcescens are urinary tract infections, pneumonia,surgical wound infections, eye infections, and bacteremia. Multiple hospital outbreaks of Serratia have been reported, with sources of infection including tap water, soap, blood transfusion products, and injected medications (2). It has also been described as a cause of endocarditis in injection drug users (3).

Serratia is intrinsically resistant to ampicillin, ampicillin-sulbactam, and 1st and 2nd generation cephalosporins due to an inducible, chromosomal AmpC beta-lactamase. Resistance to later-generation cephalosporins may be induced through exposure to these antibiotics, despite not being detected on initial antibiotic susceptibility tests. Thus, susceptibility testing is misleading and thirdgeneration cephalosporins (such as ceftazidime, ceftriaxone, and cefpodoxime) should be avoided for the treatment of Serratia species regardless of in vitro susceptibility.

A 54 year old male presented in the emergency room with worsening of shortness of breath and chest pain. He has a history of a bicuspid aortic valve that was treated with bio-prosthetic aortic valve replacement seventeen years ago and a second aortic valve replacement seven years ago. The patient’s echocardiogram showed severe aorticstenosis and moderate to severe mitral regurgitation. During hospital stay he started to show signs of low cardiac output syndrome and an intra-aortic balloon pump was placed. During sternotomy for aortic valve replacement and mitral valve repair they discovered a severely calcified and stenotic valve with additional debris that could be consistent with endocarditis. Tissue culture was sent.

Gram stain showed pink strings that could be gram negative rods, but could also be tissue debris due to tissue grinding (Image 1). After 3 days of incubation, some colonies grew on 5% sheep blood (Image 2) and chocolate agar plates with no growth on MacConkey selective medium.

Colony Gram stain made from these colonies (Image 3) was compared with the initial gram stain and showed similar type of pleomorphic gram negative rods. MALDI-TOF identified this organism as Cardiobacterium hominis.

Cardiobacterium hominis is a fastidious, pleomorphic, non-motile, gram negative bacillus and member of the HACEK group which comprises Haemophilus species, Aggregatibacter, Cardiobacterium hominis, Eikenella corrodens, and Kingella kingae. C. hominis is present as normal flora of the oropharynx in most individuals but it has also been attributed to cause infective endocarditis.

C hominis is a fastidious bacterium that grows best in the presence of increased levels of CO2 and high levels of humidity and often takes several days to grow on solid media (1). It can be distinguished from other HACEK members by a positive oxidase reaction, the production of indole and the absence of catalase activity and nitrate production.

Some of the risk factors leading to C hominis endocarditis include dental work, structural cardiac abnormalities, previous valve replacement, dilated cardiomyopathy and past history of rheumatic heart disease and endocarditis (2). The illness usually follows a sub acute course with symptoms lasting for weeks or months (1). Patients will often report fever, myalgia, anorexia, and weight loss. C. hominis tends to form large, friable vegetations associated with cerebral embolization or mycotic aneurysm formation and this might be responsible for atypical presentation of endocarditis(1). The overall prognosis of endocarditis due to C. hominis is quite favorable, despite the frequent need fo rvalve replacement (3).

Third generation cephalosporin (ceftriaxone) is considered the drug of choice for C. hominis endocarditis. Ampicillin can be used after susceptibility testing. Ampicillin-sulbactam or ciprofloxacin are alternative therapeutic options.

The patient is a 21 year old male with a history of developmental delay and chronic kidney disease secondary to posterior urethral valves, status post kidney transplant at age 14, who presents for a routine office visit with his pediatric nephrologist. In the past year, he has had chronic antibody-mediated transplant rejection despite immunosuppression. In addition, he drinks 1-1.5 gallons of water daily, self-catheterizes every three hours, and has an indwelling Foley at night. During the office visit, he denies any urinary symptoms, including dysuria, hematuria, cloudy urine, reduced output, or fever. However, given his significant risk factors for urinary tract infection, his provider orders a urinalysis and urine culture.

Laboratory Identification

The urine was noted to be cloudy, was positive for nitrites and leukocyte esterase, and had 11-50 white blood cells per high-powered field.

Urine culture demonstrated the growth of two organisms, one of which was identified to be greater than 100,000 CFU of Proteus miribalis, and the second of which grew 10,000-100,000 CFU, was isolated, and is shown below:

Mass spectrometry by MALDI-TOF confirmed that this second organism is Streptococcus pneumoniae, a bile-soluble gram positive diplococci.

Discussion

S pneumoniae is implicated in a number of diseases, but it is an uncommon pathogen in the urine. Several case-series and case reports have been published demonstrating a predilection of pathogenic urinary S pneumoniae for pediatric patients with urinary tract abnormalities. In one series, 26 urine cultures from 18 patients were identified as growing S pneumoniae, with CFU counts ranging from 100 to 100,000. Sixteen of the 26 cultures grew only S pneumoniae. Of the 18 patients, only six were adults, eight had had a kidney transplant, and four others had chronic problems with their kidneys (1). In another series of three pediatric cases, one patient had congenital bilateral duplication of the renal collecting system, one had a “congenital imperforate anus (high type 1A) with a rectovesical fistula and grade 4 bilateral vesicoureteral reflux,” and the third had bilateral renal dysplasia (2). Neither case series was able to identify a specific serotype of S pneumoniae responsible for these infections.

As discussed by Choi et al, the altered flow dynamics of the abnormal urinary systems in these patients may be compromising normal host immune clearance mechanisms, thereby increasing the susceptibility to infection (2, 3). However, it is unclear why S pneumoniae infections have a predilection for congenital urinary tract abnormalities, as opposed to all urinary tract abnormalities. Choi et al postulate that some of the gene polymorphisms known to predispose individuals to UTI or pneumococcal infections could be genetically linked to genes responsible for urinary tract abnormalities, thus increasing the probability that an individual with a congenital urinary tract abnormality would have an S pneumoniae urinary tract infection (2,4).

Given the patient’s history and risk factors, the presence of S pneumoniae in his urine was found to be significant. Treatment of both organisms and appropriate follow-up was recommended.

A 44 year old male presented to the emergency department with severe, throbbing back pain in his mid-thoracic spine. He states the pain began a couple weeks ago and noted no recent fevers or night sweats, but does admit to chills. His past medical history is significant for end stage renal disease requiring dialysis, insulin dependent diabetes mellitus, and multiple amputations. On physical examination, there was tenderness to palpation along spine in mid-thoracic region. Lab work showed a normal white blood cell count, C reactive protein of 0.90 mg/dL (0.00 – 0.50 mg/dL), and an erythrocyte sedimentation rate of 60.0 mm/hr (0.0 -10.0 mm/hr). MRI of the spine was consistent with discitis and osteomyelitis at T7-8 with compression fractures causing spinal stenosis and cord compression. Given the concern for an infection process, blood cultures were collected and interventional radiology performed a bone biopsy. The specimen was sent for bacterial, fungal, and AFB cultures as well as for histology.

The organism grew as discrete, creamy colonies growing on blood agar and Sabouraud dextrose agar after 48 hours of incubation at 35°C and resembled a yeast. MALDI-TOF mass spectrometry identified the isolate as Candida parapsilosis. Similarly, the surgical pathology specimen showed necrotic bone with inflammation and yeast forms and pseudohyphae consistent with a Candida spp. infection. Blood cultures were negative. On chart review from an outside hospital, it was discovered the patient had an episode of candidemia ten months ago which was thought to be related to his dialysis port.

Discussion

Yeasts are ubiquitous in the environment and make up the normal microbiota of human skin as well as the oral cavity, gastrointestinal tract and genitourinary tract. In general, when Candida spp. cause infections it is thought to an opportunistic infection acquired endogenously and due to exposure to prolonged antibiotics, suppressed immune system, or as a result of intravascular catheters. Those with diabetes mellitus, mucositis, bowel perforations, and intravenous drug users are most susceptible. Infections with Candida parapsilosis are becoming more common, and have the potential to cause invasive disease, such as fungal endocarditis and severe infections in the neonatal population.

In the microbiology laboratory, C. parapsilosis grows rapidly as discrete, creamy colonies on a variety of agars. On cornmeal-Tween 80 agar, C. parapsilosis grows as short, curved pseudohyphae with blastoconidia arranged singly or in small clusters at points of constriction. The arrangement is sometimes described as resembling a sage bush. C. parapsilosis is germ tube negative and is negative for urease. In many laboratories currently, identification is achieved by automated methods, such as Vitek 2, or mass spectrometry, allowing for more rapid and accurate identification.

Anti-fungals, such as echinocandins, azoles, and amphotericin B, are all potential therapeutic options to treat C. parapsilosis infections. CLSI C.parapsilosis specific breakpoints exist for fluconazole, voriconazole,micafungin, caspofungin, and anidulafungin in the M27-S4. Susceptibility testing should be performed on significant isolates from normally sterile sites.

In the case of our patient, infectious disease was consulted and he was started on IV micafungin and then transitioned to oral fluconazole. He had a transesophgeal echo and eye exam performed to ensure he didn’t have endocarditis or an invasive eye infection due to hematogenous spread of the yeast. He was discharged home on long term oral fluconazole.

-Rim Alkawas, MD, is a second year Anatomic and Clinical Pathology resident at the University of Mississippi Medical Center.

-Lisa Stempak, MD, is an Assistant Professor of Pathology at the University of Mississippi Medical Center in Jackson, MS. She is certified by the American Board of Pathology in Anatomic and Clinical Pathology as well as Medical Microbiology. She is the Director of Clinical Pathology as well as the Microbiology and Serology Laboratories. Her interests include infectious disease histology, process and quality improvement, and resident education.

A young adult female presents to an urgent care clinic with the chief complaint of a “bump and surrounding redness” on her right medial thigh. The patient reports the bump had been present without change for 1 year. Approximately 2 days prior to presenting at the urgent care clinic the patient states she nicked the bump while shaving, and subsequently the bump became tender with surrounding erythema and produced purulent drainage. The patient denies any similar prior lesions and denies any significant past medical history. On physical exam, the lesion measured 1 cm with the surrounding erythema measuring 5cm. The urgent care physician performed an incision and drainage and noted a scant amount of white purulent material within the lesion. A cyst wall was identified and was removed as much as possible. A swab of the purulent material was collected and submitted to microbiology for culture.

Laboratory Identification

The primary gram smear of the swab specimen was interpreted as no bacteria or polys seen. Routine culture media including blood, chocolate, MacConkey, and CNA agar were inoculated and incubated aerobically. Following incubation, the blood agar showed few gram positive cocci consistent with usual skin flora and few single morphology of medium to large sized gray colonies without hemolysis. On the MacConkey agar, few single morphology non-lactose fermenting colonies were identified. The gray colonies identified on the blood agar gram stained as gram negative bacilli with unremarkable morphology. An oxidase test was performed and the bacteria was found to be oxidase positive. The key biochemical and physiologic characteristics of the isolate included: good growth on thiosulfate citrate bile salts and sucrose (TCBS) agar with yellow colonies, good growth in 6% NaCl nutrient broth, and no growth in 0% NaCl nutrient broth. The organism was identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) as Vibrio alginolyticus.

Image 1. Blood agar isolate of medium sized gray colonies.

Image 2. MacConkey agar with non-lactose fermenting colonies.

Discussion

Vibrio spp. are water organisms commonly found in marine or brackish water environments. These organisms are gram negative bacilli which classically have “comma” shaped morphology on gram smear, though this is not an absolute. On sheep blood agar, these organisms are variably beta hemolytic medium to large gray colonies and on MacConkey agar are non-lactose fermenting (with the exception of Vibrio vulnificus). Vibrio spp. are oxidase positive, ferment glucose, and readily grow on most isolation media with growth being enhanced with the addition of 1% NaCl to the media. The growth characteristics on media containing different concentrations of NaCl can be used in differentiating the different Vibrio spp. Thiosulfate Citrate Bile Salts and Sucrose (TCBS) agar is both selective and differential for Vibrio spp. with sucrose fermentation being detected as yellow colony formation.

Vibrio angiolyticus typically causes extraintestinal infections, with wound infections and otitis externa being the most frequent. Transmission frequently occurs via traumatic aquatic injuries in contaminated water. Vibrio angiolyticus rarely causes intestinal disease and is isolated in less than 5% of stool cultures in patients with Vibrio associated diarrhea. Growth characteristics of Vibrio alginolyticus include yellow colonies on TCBS due to its ability to ferment sucrose and good growth on 6% NaCl and no growth on 0% NaCl. Additional key biochemical characteristics of Vibrio alginolyticus include oxidase positivity, nitrite positivity, negative for myo-Inositol fermentation, negative for arginine dihydrolase, positive for lysine decarboxylase, and variable positivity for ornithine decarboxylase. Most wound infections due to Vibrio alginolyticus are non-severe, and most mild infections will clear without antibiotic therapy.

A 72 year old female with past medical history of stage IV ovarian adenocarcinoma treated with chemotherapy and interval debulking surgery, presented to emergency room with a one week history of confusion and worsening balance.

CT scan of the head showed new communicating hydrocephalus. A magnetic resonance imaging couldn’t be performed initially because of patient’s uncontrolled agitation. Lumbar puncture (LP) was performed. Following this procedure the patient’s mental status showed some improvement and therefore neurosurgery team decided to insert ventriculoperitoneal (VP) shunt to treat her hydrocephalus and prevent recurrence of seizures.

It was Friday afternoon when a microbiology technologist brought the patient’s cerebrospinal fluid (CSF) gram stain to be reviewed. It was confirmed that no inflammatory cells and organisms were present. However, cells in the background looked very atypical (Image 1a, b).

The gram stain is used to provide preliminary information about the microorganism present in the specimen. Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be “Gram-positive,” whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be “Gram-negative” (1). An adequate examination of a gram-stained smear includes observing numerous representative fields and the fields containing neutrophils yield the most information (2). Gram stain provides information about number of bacteria present, gram reaction and shape of the bacteria. In background we can also see epithelial cells and inflammatory cells. However, it’s a good practice to also appreciate and investigate any odd looking findings.

To investigate further, we visited the hematology laboratory to view their CSF slide to determine if these cells were a processing artifact. After it was confirmed that hematopathology saw the same atypical cells, a cytopathologist was requested to review the gram stain since patient’s CSF cytology specimen was to be processed after the weekend. Cytopathologist favored our suspicion and decided to process the cytology specimen late in the day on Friday and it was confirmed that those atypical cells were consistent with the metastatic adenocarcinoma.

Neurosurgery team was immediately contacted to reconsider insertion of the VP shunt as the shunt would drain fluid from the CSF into the peritoneal cavity and thus there was concern for transferring of malignant cells from central nervous system into abdomen/pelvis. However, after consulting oncology team it was later decided to proceed with the procedure since patient’s primary tumor originated in abdomen/pelvis and current intraabdominal tumor burden was not significant as compared to the symptoms driven by CNS involvement. Proceeding with this procedure was considered to be palliative and the best course of action to improve the patient’s quality of life.

A 64 year old man with metastatic colon cancer and a history of recent motor vehicle collision with polytrauma presented from a rehabilitation facility with fever up to 105 degrees Fahrenheit. Two months prior to admission he was hospitalized for the motor vehicle collision in which he sustained orthopedic injuries to multiple extremities. In addition to external fixation of several injuries, he returned to the operating room on multiple occasions for additional incision and drainage of a wrist wound which demonstrated gross purulence with cultures growing Enterococcus, Prevotella, and an extended spectrum beta-lactamase-producing Morganella morganii. Antimicrobial regimen initially consisted of surgically placed antibiotic beads and broad-spectrum therapy with vancomycin, piperacillin-tazobactam, and then meropenem. The patient was eventually transitioned to an oral antibiotic regimen consisting of linezolid and ciprofloxacin for an anticipated course of six weeks and he was discharged to a rehabilitation facility.

After several weeks at the rehabilitation facility, the patient became febrile and was admitted for workup of his fever. Initially the fever was of uncertain origin with malignancy (rectal cancer with metastasis to the liver), drug fever (linezolid), and wound site infection on the differential. Linezolid was discontinued, daptomycin initiated, and ciprofloxacin maintained. Fever persisted and ciprofloxacin was discontinued as another possible source of drug fever. Ertapenem was initiated. Initially, prior wounds and surgical sites appeared well-healing. Blood cultures all yielded no growth. However, on day five of this hospitalization, purulent drainage was noted from the site of a left leg surgical wound. Arthrocentesis yielded 0.5 mL of bloody fluid which was sent for cell count, differential, and culture.

Laboratory Findings

Initial Gram stain showed few polymorphonuclear leukocytes with no bacteria seen. Cell count was unable to be performed due to viscosity of the specimen but differential showed 80% neutrophils. There was no growth on aerobic blood, chocolate, or MacConkey agars. Anaerobic Schaedler (non-selective) agar grew 1-2 mm brownish colonies (Image 1). Gram stain of this isolate revealed gram variable bacilli forming long filaments (Image 2). The isolate was identified using MALDI-TOF MS (Vitek) as Clostridium ramosum.

The patient was taken to the operating room for incision and drainage of the left knee with two additional samples sent for culture which grew Clostridium ramosum.

Discussion

Clostridium species are anaerobic, spore-forming, gram positive bacilli. C. ramosum is non-motile and is normally found in the human colon and the environment. One study identified C. ramosum in the feces of 83% of sampled adults. Former names include Eubacterium filamentosum, Ramibacterium ramosum, Actinomyces ramosus, and Eubacterium ramosum. Figure 2 demonstrates a notable characteristic of C. ramosum, i.e. its variable appearance on Gram stain. The morphology here may be described as gram negative or “over-decolorized”, though gram positive bacilli are clearly seen forming many of the filaments. Its terminal endospores are often difficult to identify on Gram stain and this is true of Figure 2. These characteristics on Gram stain have historically made identification difficult, though use of MALDI-TOF MS facilitated identification in our case. Biochemically, C. ramosum ferments glucose and hydrolyzes esculin; it is negative for lecithinase and lipase.

C. ramosum possesses an IgA protease though it is not commonly pathogenic. When it is pathogenic, the spectrum of disease overlaps with that of other anaerobes and includes deep-seated abscesses, e.g. intra-abdominal abscess secondary to trauma. Osteomyelitis and primary bacteremia are also possible, particularly in immunocompromised patients. Otitis media in children is another possible clinical scenario.

Septic arthritis due to Clostridium ramosum

A 2016 case report described two cases of septic arthritis due to C. ramosum. In one case, a patient with rheumatoid arthritis on methotrexate and prednisone and history of revision knee arthroplasty eight years prior presented with knee swelling. Synovial fluid aspirate was consistent with an infectious process; the prosthesis was removed but synovial and intraoperative cultures were negative. Antimicrobial therapy with linezolid and ciprofloxacin was administered for six weeks with clinical improvement. Two weeks after discontinuation of antibiotics the patient became febrile. Blood cultures were negative but culture of synovial fluid grew C. ramosum. The patient required multiple operations due to joint destruction and was ultimately managed with intravenous penicillin and clindamycin with transition to oral metronidazole for three months of therapy. The second case of C. ramosum septic arthritis presented in this report was ultimately managed with surgical debridement and amoxicillin-clavulanate. Both cases presented in patients with immunocompromising comorbidities and the course of their septic arthritis was chronic, recurring, and destructive but non-fatal with both patients dying from other causes.

These clinical and laboratory characteristics are consistent with the case of C. ramosum septic arthritis identified at our institution. The case of septic arthritis presented here involved an immunocompromised host (malignancy) with history of trauma, foreign body placement (external fixator), and long-term antibiotic therapy. This patient’s wound required debridement in the operating room on three occasions. Once clinically stable, the patient was discharged to a subacute rehabilitation facility and continued on ertapenem with amoxicillin for an expected duration of six weeks with the plan to switch to amoxicillin-clavulanate and ciprofloxacin for suppressive therapy.